The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

OBJECTIVEThe characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes.RESEARCH DESIGN AND METHODSClinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01–31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04–0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed.RESULTSIA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec–derived peptides to elicit CD4+ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec.CONCLUSIONSWe developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.

Autoantibodies directed against tyrosine phosphatase IA-2 antibody (IA-2 Ab) are diagnostic for autoimmune type 1 diabetes. Conventional assays target the intracellular domain of IA-2. Among patients with ketosis-prone diabetes (KPD), characterized by presentation with diabetic ketoacidosis (DKA), >60% of adults lack three classic islet autoantibodiesdIA-2, GAD65, and ZnT8 Absdassociated with type 1 diabetes. We aimed to determine whether apparently autoantibodynegative ("A2") KPD patients possess occult IA-2 Ab directed against fulllength IA-2 (IA-2FL) or its extracellular domain (IA-2EC). RESEARCH DESIGN AND METHODS We developed an assay that targets IA-2FL and IA-2EC and used it to analyze 288 subjects with A2 KPD. RESULTS Ten A2 KPD patients were positive for IA-2EC Ab (3.5%), and three were also positive for IA-2FL Ab (1.0%), similar to frequencies in type 1 and type 2 diabetes. CONCLUSIONS Measurement of IA-2FL Ab and IA-2EC Ab improves the accuracy of the Ab classification of KPD patients. Ketosis-prone diabetes (KPD) is a heterogenous syndrome characterized by presentation with diabetic ketoacidosis (DKA) and classified by the presence or absence of islet autoantibodies ("A+" or "A2") and presence or absence of b-cell functional reserve ("b+" or "b2") (1,2). Distinct from patients with type 1 diabetes, patients with KPD often present when older, have fewer recurrences of DKA, and can often discontinue insulin treatment while maintaining glycemic control (3). More than 60% of KPD adult patients lack evidence of islet autoimmunity (i.e., are A2) by testing for the presence of autoantibodies against the 65-kDa isoform of glutamate decarboxylase (GAD65), zinc transporter T8 (ZnT8), and the neuroendocrine autoantigen IA-2 (or ICA512) (1,2,4). Constructs used in conventional IA-2 autoantibody assays include intracellular fragments, but not the extracellular domain (IA-2EC), which has recently been investigated as a target for IA-2-specific autoantibodies (5). We reported that 1% of patients with autoimmune type 1 diabetes are positive only for the IA-2EC antibody (Ab), as were 4.7% of 258 patients with type 2 diabetes (5,6). Furthermore, we reported that full-length IA-2 (IA-2FL

We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys 27 , Gly 608 , and Pro 671) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2varspecific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the threedimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as "low risk" relatives. Type 1 diabetes (T1D) is an autoimmune disease that results from the targeted destruction of pancreatic b-cells by autoreactive T cells (1,2). The development of T1D is associated with the occurrence of autoantibodies (AAb) to pancreatic islet antigens that can be used as predictive biomarkers of disease progression (3). AAb associated with T1D are mainly directed against proteins that are involved in the secretory pathway of insulin, including insulin, glutamic acid decarboxylase (GAD65), islet tyrosine phosphatase-like protein (IA-2), and zinc transporter 8 SLC30A8 (ZnT8). The presence of AAb to IA-2 is associated with a high risk of T1D development (4-7). Screening for T1D-associated AAb allows for identification of asymptomatic, high-risk individuals (8) and for natural history studies of disease in cadaveric donors (9). The neuroendocrine molecule IA-2 is a transmembrane glycoprotein of the tyrosine phosphatase-like protein family that is localized to the insulin-secretory granules of the pancreatic b-cell (10). IA-2 (PTPRN) encodes a 979-amino acid protein containing three domains: the N-terminal extracellular (or luminal) domain (amino acids 1-556), the transmembrane domain (amino acids 557-600), and the COOH-terminal intracellular (or cytoplasmic) domain (amino acids 601-979) containing a juxtamembrane (JM) domain (amino acids 601-686) and a protein tyrosine phosphatase

OBJECTIVE Continuous glucose monitoring (CGM) parameters may identify individuals at risk for progression to overt type 1 diabetes. We aimed to determine whether CGM metrics provide additional insights into progression to clinical stage 3 type 1 diabetes. RESEARCH DESIGN AND METHODS One hundred five relatives of individuals in type 1 diabetes probands (median age 16.8 years; 89% non-Hispanic White; 43.8% female) from the TrialNet Pathway to Prevention study underwent 7-day CGM assessments and oral glucose tolerance tests (OGTTs) at 6-month intervals. The baseline data are reported here. Three groups were evaluated: individuals with 1) stage 2 type 1 diabetes (n = 42) with ≥2 diabetes-related autoantibodies and abnormal OGTT; 2) stage 1 type 1 diabetes (n = 53) with ≥2 diabetes-related autoantibodies and normal OGTT; and 3) negative test for all diabetes-related autoantibodies and normal OGTT (n = 10). RESULTS Multiple CGM metrics were associated with progression to stage 3 type 1 diabetes. Specifically, spending ≥5% time with glucose levels ≥140 mg/dL (P = 0.01), ≥8% time with glucose levels ≥140 mg/dL (P = 0.02), ≥5% time with glucose levels ≥160 mg/dL (P = 0.0001), and ≥8% time with glucose levels ≥160 mg/dL (P = 0.02) were all associated with progression to stage 3 disease. Stage 2 participants and those who progressed to stage 3 also exhibited higher mean daytime glucose values; spent more time with glucose values over 120, 140, and 160 mg/dL; and had greater variability. CONCLUSIONS CGM could aid in the identification of individuals, including those with a normal OGTT, who are likely to rapidly progress to stage 3 type 1 diabetes.

changes in the functions of regulatory B cells over time and a potentially novel mechanism for inducing the suppressive capabilities of regulatory B cells using IL-5-induced IL-10 production. Results Diabetes is significantly delayed in an adoptive transfer model following injections of MHC-compatible CD19 + cells from young donor NOD mice. Splenocytes isolated from MHC-compatible diabetic female NOD mice were i.v. injected into 6-week-old NOD.scid recipient female mice. NOD.scid recipients receiving single transfers of diabetic splenocytes started to develop T1D at day 20 after transfer (Figure 1A). Cotransfer experiments were performed on day 6 and day 12 using CD19 + cells purified from 6-week-old prediabetic female NOD mice to create a boosted B cell pool mimicking the young prediabetes phase of the NOD donor. We observed a strikingly significant delay in progression to autoimmune diabetes in NOD.scid recipients when purified splenic CD19 + cells from 6-week-old NOD mice were cotransferred (Figure 1A; P < 0.0001). By day 40 after transfer, 100% of the NOD.scid recipients receiving diabetic splenocytes alone had progressed to overt diabetes, while 100% of NOD.scid CD19 + cotransfer recipients were still normoglycemic (Figure 1A). CD4 + and CD8 + T cell populations (gated initially on CD3 + CD19-) were not significantly different after the reconstitution process in NOD.scid recipients receiving either NOD splenocytes alone or CD19 + cotransfers (Figure 1B). Further analysis of the B cells from 6-week-old NOD female mice and matching C57BL/6 and Balb/c controls found that NOD mice have an increased number of CD19 + IgM + CD5 hi CD1d lo traditionally described as Bregs in NOD mice as compared with control strains (Figure 1C) (34-37). Analysis of the CD3 + CD4 + Th repertoire within the spleen revealed a normal distribution of Th1 (IFN-γ-secreting) and Th17 (IL-17A-secreting) T cells, with most of the T cells in the spleen of both NOD.scid recipient populations containing a majority of Th1 pool (Figure 1D), as established in the literature (38, 39). To investigate the possible effect of age, cotransfer experiments were executed by using splenocytes from diabetic NOD donors combined with CD19 + B cells from either 6-week-or >15-week-old nondiabetic female NOD mice (15). While a similar delay in onset as the previous experiment was observed when CD19 + cells from young donors were cotransferred, NOD.scid recipients of CD19 + cells from >15-week-old nondiabetic NOD donors had a similar rate of diabetes progression compared with recipients of splenocytes alone obtained from NOD diabetic donors (Figure 2A). These are the first observations to our knowledge demonstrating that >15-week-old NOD female donors can transfer diabetes within the same amount of time as an already-diabetic NOD female donor. Further analysis of the CD19 + B cell pool taken from the spleen (Figure 2B) found an increased number of antigen-presenting capable marginal zone (MZ) B cells (48.4%) and MZ precursors (16.7%) as a percentage of the total CD...

We previously demonstrated that the IA2 variant (IA2var) autoantibody (AA) is associated with enhanced prediction of type 1 diabetes (T1D) in at-risk individuals but its effect on progression through preclinical T1D stages is unknown. We aimed to assess the influence of IA2var AA on rates and risks of transitions from Stage 1 (multiple AA positive, no dysglycemia) to Stage 2 (multiple AA positive, dysglycemia) and to Stage 3 (clinical T1D). Participants in the Diabetes Prevention Trial (DPT-1) with Stage 1 or Stage 2 T1D at baseline and having ≥2 glucose tolerance tests during follow-up were selected (N=175). Standard AA (GAD65, ICA, ICA512 and insulin AA) were considered as biomarkers in addition to IA2var. Data analysis used Markov transition models. The estimated 12-month probability of transition from Stage 1 to 3 (N=89) was 13.8% (95% CI: 9.7%, 18.8%) and from Stage 2 to 3 (N=86) it was 41.3% (30.6%, 52.6%). IA2var AA, sex, age and were statistically significant risk factors for the Stage 2 to 3 transition (Table 1, B). Importantly, IA2var AA (the only significantly influential AA) was associated with a reduction in the risk of transition from Stage 2 to 3. In sum, male sex, older age and positive IA2var AA significantly decreased the risk of transition from Stage 2 to Stage 3 T1D. This information has important implications for the selection of candidates for T1D prevention in clinical and research practices. Disclosure C.Beam: None. S.Pietropaolo: None. M.J.Acevedo-calado: None. M.A.Herman: Research Support; Eli Lilly and Company. M.J.Redondo: None. M.Pietropaolo: None. Funding National Institutes of Health (2R01DK053456-19A1); Robert and Janice McNair Foundation

Autoimmune disorders such rheumatoid arthritis (RA) harbor autoantibodies against a variety of post-translationally modified (PTM) proteins. We aimed to investigate whether PTM epitopes of IA-2, a major autoantigen related to type 1 diabetes (T1D), are targeted by autoantibody responses. The extracellular domain of IA-2 (IA-2-ec) was synthesized incorporating deamidated amino acid residues (IA-2ec-PTM) previously found to elicit T cell responses in T1D patients. IA-2ec-PTM was then subjected to immunoprecipitation with sera from T1D patients. Notably, autoantibody reactivity towards IA-2ec-PTM was present in 28% of T1D patients as compared to ~9% of serum reactivity against the native IA-2ec. The area under the ROC curve (ROC AUC) of IA-2ec-PTM autoantibodies revealed that these autoantibodies can adequately distinguish between T1D patient and control groups (ROC AUC: 0.7132; P &lt; 0.0001). Relatives of T1D probands (from the TrialNet Pathway to Prevention Study) carrying both IA-2ec-PTM AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progressed rapidly to onset of T1D (Stage 3) compared to those relatives who did not carry these immunologic abnormalities (P = 0.003). Understanding the mechanisms underlying PTMs of islet autoantigens, such as IA-2ec-PTM, is critical to develop new research to determine the ability of modified epitopes to induce islet autoimmunity, their potential role as biomarkers of islet cell injury as well as breaking immune tolerance to islet cell antigens through neo-antigen formation. Disclosure M.J. Acevedo-Calado: None. S. Pietropaolo: None. L. Yu: None. A.W. Michels: Stock/Shareholder; Self; IM Therapeutics. M. Pietropaolo: None. Funding National Institutes of Health